The immune system has several different mechanisms for dealing with pathogens in the system (Parker, D. C. 1993. Annu. Rev. Immunol. 11:331-360; Clinical Immunology: Principles and Practice. Vols. 1 and 2. eds (Fleisher et al. 1996. Mosby-Year Book, Inc. New York, N.Y.). The first step in the immune response is the activation of a special subclass of T lymphocytes called helper T cells. Macrophages present fragments of foreign proteins, or antigens, on their surfaces. Recognition of these antigens by specialized receptors found on helper T cells then initiates the two responses: a cell-mediated immune response and a humoral immune response.
The cell-mediated response involves principally the stimulation of another subclass of T lymphocytes called cytotoxic T lymphocytes (CTLs) that recognize and destroy infected cells. HLA class-I restricted molecules bind to peptides that have been processed intracellularly and enable CD8+T cells to eliminate intracellular viruses. CTLs are the second line of immune defense since the humoral response generates antibodies that may be able to neutralize virus and decrease the number of infected cells from the original infection. The CD4+T (Th) cells often have a helper phenotype, which is measured by their ability to assist the humoral response. The cells function to facilitate the effector function of other cells, and while Th cells have limited antiviral activity, they play a major role in providing intermolecular cooperation with antibody-producing B cells (Vitetta et al. 1989. Adv. Immunol. 45:1; Hodes, R. J. p. 587 In: 1989. Fundamental immunology, eds Paul, W. E.).
The humoral response, on the other hand, involves the activation of the second major class of lymphocytes, the B cells, to produce circulating antibodies. Antibodies recognize and neutralize soluble antigens, and mark cells or viruses bearing antigens for destruction by phagocytic cells. The HLA class-II molecules present the exogenous antigenic peptides to the class-II restricted CD4+B cells. Antibodies serve as efficient means of reducing the viral infectivity by several mechanisms. The antibody response to surface antigens on an invading pathogen can be very strong, and the antibody may be able to decrease the titer of virus particles significantly. In addition, antibodies can alter the structural integrity of the invading pathogen through interaction with surface antigens, which can make the virus non-infectious. Neutralization of the pathogen can also occur by preventing interaction with the cellular receptor and/or preventing endocytosis into the cytoplasm (Ruggeri, F. M. and Greenberg, H. B. J. Virol. 1991;2211-2219)Dimmock, N.J. In: Current Topics in microbiology and immunology. 1993. Springer-Verlag: New York).
Development of effective vaccines has been one of the most decisive advances leading to the dramatic downward trend in incidence of viral diseases. Vaccination induces a "primed" state in the vaccinated subject so that, following exposure to an antigen, a rapid secondary immune response is generated leading to accelerated elimination of the organism and protection from clinical disease. Designing vaccines requires attention to the safety of the system as well as to the antigenicity and efficacy of the prophylaxis.
It has been known for some time that humoral and cell-mediated responses to antigens can be quite different. In general, B cell epitopes of antigens are longer, and are known as conformational epitopes. Conformational epitopes require the proper 3-dimensional structure for efficient recognition by antibodies (Elner et al. 1977. J. Immunol. 118:2053). In contrast, T cells usually recognize small linear epitopes based on sequential information. Since effective resistance to viral or bacterial infection requires the activities of both humoral and cellular components, it is important to optimize the presentation of antigens to both B and T cells.
Despite recent advances in epitope presentation systems, there is still a need in the art for a system that is genetically capable of expressing of B and T cell epitopes concurrently, as well as presenting these epitopes to the immune system in the proper context.
There is evidence that simultaneous expression of T and B cell epitopes on the same carrier system can enhance the antibody response through a cooperative mechanism between B and T cells. For example, enhanced B cell vaccines to Hepatitis B are possible by stimulating hepatitis B virus (HBV) envelope protein-specific B cells though a carrier system that mimics HBV (Chisari, F. V. 1995. pp. 29-60 In: Annu. Rev. Immunol). If this carrier, or mimic, carries Th cell epitopes, the subsequent processing of these Th epitopes can induce the proliferation of B cells. For example, the HBV clearance depends on a vigorous and polyclonal B cell and T cell response to the envelope, nucleocapsid, and polymerase antigens. While the antibody responses to the HBV nucleocapsid and polymerase antigens are not well understood, it is quite clear that the antibody response to HBV envelope antigen requires T-cell cooperation. Despite the need for the vigorous immune response necessary to clear HBV, the class-II restricted envelope-specific response is quite low in acute and chronic hepatitis. The processing of Th epitopes can induce the proliferation of B cells and anti-envelope antibodies. An efficient means of enhancing the cooperation between B and Th cells due to increasing the exposure of B and Th cell epitopes via a carrier system would likely result in disease remission in patients. There is evidence that enhanced B cell vaccines to Hepatitis B are possible by stimulating HBV envelope-specific B cells though a carrier system that mimics HBV by properly presenting HBV determinants to antigen presenting cells (APCs).
While there is a substantial response of Class I-restricted CTL in acute cases, the CTL response to HBV is not easily detected in chronically infected patients who have been unable to clear virus. The ability to induce vigorous CTL expansion along with B and Th cell cooperation could have profound significance for the treatment of HBV infection as well as many other infectious diseases and cancer.
There have been remarkable advances made in vaccination strategies recently, yet there remains a need for improvement on existing strategies. Recombinant pathogenic viruses have often induced the strongest humoral and cell-mediated responses, but the issues of safety and potential interference with pre-existing immunity to the vectors have not made them an attractive system for continued use. Peptide vaccines have been shown to be relatively safe with a strong monofunctional immune response. Despite these advantages, they tend to be poorly immunogenic and have a limited ability to bind selectively to different MHC determinants found in genetically distinct populations of individuals. While it may be possible to produce numerous peptides for formulation into a single vaccine, such an undertaking presents a formidable task. Genetic immunization, or DNA vaccines, has shown promise, but has not shown the ability to target APCs and produce a broad polyclonal response. Despite these advances, the current technologies have not provided a system that was competent in the simultaneous expression of B and T cell epitopes that effectively prime B and T cells of the immune system.
In these non-limiting examples, genetically-engineered Nodaviral or specifically the Flock House Virus (FHV) chimeric coat protein constructs were made to include well-defined ligands within the insertion region as well as encapsidation of therapeutic genes or antisense.
Gene delivery or gene therapy can be defined as the delivery of a functional gene (for expression of a protein) or an antisense molecule (for blocking translation of a protein) to somatic cells. See, for example, U.S. Pat. No. 5,589,466 to Felgner et al. and U.S. Pat. No. 5,676,954 to Brigham. For reviews see Mitani, K, and Caskey, C. T. (1993) TIBTECH 11:162-166, Findeis, M. A. et al. (1993) TIBTECH 11:202-205; Friedmann, T. (1994) TIG: 10:210-214, Smith, C. (1994) TIG: 10:139-144; Karpati et al. (1996) TIBS 19:49-54; Calos, M. P. (1996) TIG: 12:463-466. Several gene delivery technologies that are being used to treat a variety of diseases and acquired and genetic disorders are summarized in Table 1.
TABLE 1 Comparison of Gene Delivery Technologies Trans- duction Insert effi- Vector Size Integration ciency Advantages Disadvantages Retrovirus 8 Yes High Stable Infects only kB transfection rapidly divid- of dividing ing cells. Can cells. be oncogenic. Adenovirus 7.5 No High Transfects Transient kB nearly all expression cell types triggers im- dividing or mune response, nondividing. common human virus Adeno 4 Yes. High Stable Small insert Associated kB (chr. 19) transfection. size, integra- Virus (AAV) tion poorly understood. Helper virus required. Herpes 20 No Low Large insert Transient Simplex kB size. Neuron expression, Virus (HSV) specific. potential to generate infectious HSV in humans Vaccinia 25 No High Infects a Limited to non- kB variety of smallpox cells vaccinated or effectively. immuno- compromised individuals. Liposomes 20 No N/A Large insert Transient kB size. expression is disadvantage combined with variable delivery. Ballistic 20 No N/A Large insert Requires ex- ("biollistic") kB size. posed tissue. Injection Plasmid 20 No N/A Large insert Poor delivery. DNA kB size. Only sustained Injection expression in muscle.
Recombinant Flock House virus (FHV) proteins displaying viral antigens have been described (Tisminetzky, S. G. et al., FEBS Lett. 353:1-4 (1994); Scodeller, E. A., et al. Vaccine, 13:1233-1239 (1995); Buratti, E., et al., J. Immunol. Methods, 197:7-18 (1996); Schiappacassi, M., et al., J. Virol. Methods, 63:121-127 (1997); Buratti, E., et al., Clin. Diagn. Lab. Immunol., 4:117-12 (1997). See also Baralle, F. E. et al., PCT Published Application WO 96/05293 (1996). These previous attempts suffer from difficulties in forming a virus-like particle due to the deletion of amino acid residues in one or more regions of the capsid protein, however.
What is needed are recombinant nodavirus related proteins that can incorporate heterologous peptides as long as 100 amino acid residues or larger yet still be capable of self-assembly into chimeric virus-like particles.